PP2A assay

PP2A activity was determined as previously described [14, 34] using the commercially available PP2A Immunoprecipitation Phosphatase Assay Kit (17-313, Upstate Biotechnology). Briefly, protein lysates were prepared in 20 mM imidazole-HCl, 2 mM EDTA, 2 mM EGTA, pH 7.0 with 10 μg/ml each of aprotinin leupeptin and pepstatin, 1 mM benzamidine, 1mM PMSF and phosphatase inhibitors tablets (Roche, Madison, WI). 50 μg of protein was immunoprecipitated with 2 μg of anti-PP2A antibody (1D6, Upstate Biotechnology) and 50 μL of protein-A-agarose beads for 2 hrs at 4°C. Beads were washed extensively with lysis buffer, then with Ser/Thr assay buffer. Beads were then used in the phosphatase reaction for measuring dephosphorylation of the phosphopeptide (K-R-pT-IR-R) according to the manufacturer's protocol using malachite green phosphate detection solution. The level of free phosphate was normalized to total amount of PP2Ac immunoprecipitated as measured by densitometry analysis of immunoblots.